Genetic Diagnosis in Non-Obstructive Azoospermic Tunisian Men

Research Article

Austin J Reprod Med Infertil. 2015;2(2): 1012.

Genetic Diagnosis in Non-Obstructive Azoospermic Tunisian Men

Wajih Hammami1,2, Olfa Kilani1,3, Mariem Ben Khelifa1,2, Wiem Ayed1,3, Abderrezzak Bouzouita3,5, Fethi Zhioua3,4, Sonia Abdelhak² and Ahlem Amouri1,2,3*

¹Department of Histology and Cytogenetics, Institut Pasteur de Tunis, Tunisia

²University of Tunis El Manar, LR11IPT05 Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunisia

³University of Tunis El Manar, Faculty of Medicine of Tunis, Tunisia

4Department of Obstetrics and Gynecology, Aziza Othmana, University Hospital, Tunis, Tunisia

5Department of Urology, Charles-Nicolle University Hospital, Medical University of Tunis, Tunisia

*Corresponding author: Ahlem Amouri, Department of Histology and Cytogenetics, Institut Pasteur de Tunis 13, Place Pasteur de Tunis, Tunisia

Received: March 05, 2015; Accepted: May 10, 2015; Published: May 20, 2015


Purpose: Male infertility is the cause in half of all childless partnerships. Numerous factors contribute to male infertility, including chromosomal aberration and Yq microdeletions. We therefore aimed to evaluate the prevalence of genetic abnormalities among non-obstructive azoospermic (NOA) Tunisian men referred for routine cytogenetic analysis to the department of cytogenetics of the Pasteur institute of Tunis.

Methods: Karyotype analyses were performed on peripheral blood lymphocytes using R-banding for 401 NOA. Molecular diagnosis of classic Yq microdeletions was performed in 90 NOA with normal karyotypes by two multiplex PCRs using six STS markers (Sequence-Tagged Site) recommended by the EAA/EMQN (European Academy of Andrology / the European Molecular Genetics Quality Network).

Results: The overall incidence of chromosomal abnormalities was 12.22% (49/401). Out of the 49 patients with abnormal cytogenetic findings, sex chromosome abnormalities were observed in 42 (85.71%) including Klinefelter syndrome in 37 (75.5%). Structure chromosome abnormalities involving autosomes (14.28%) and sex chromosomes (2.04%) were detected in 8 infertile men. Furthermore, the Yq microdeletions were seen in two patients (2.22%). Both had complete deletion of the AZFc region.

Conclusion: The occurrence of chromosome anomalies and Yq microdeletions among NOA men strongly suggests genetic testing and counseling prior to employment of assisted reproduction techniques in Tunisia.

Keywords: Male infertility; Non-obstructive azoospermia; Chromosomal abnormalities; Y-chromosome microdeletion


Infertility is a major health problem affecting up to 15% of couples of reproductive age [1]. For many years, it was assumed that most reproductive problems could be attributed the female partner, but research in recent years has demonstrated that 30-50% of infertility is caused by male factor [2].

Until recently, there was no treatment available for men who have a complete absence of sperm in the ejaculate (azoospermia). This one accounts 10-15% of male infertility patients [3-5], among them 50-60% of which are non-obstructive azoospermia (NOA), which is characterized by the absence of sperm in the ejaculate without the obstruction of the reproductive tract pathway [6]. NOA occurs in ~1% of the male population and approximately 20-25% of NOA patients are caused by known genetic abnormalities [3, 7, 8]. Those involving chromosome anomalies to about 15%-16% [9]. The most frequent one being the 47, XXY karyotype that characterizes the Klinefelter Syndrome (KS) with a frequency 14% [9], followed by the microdeletions of the long arm of the Y chromosome (Yq) removing the azoospermia factor (AZF) region or parts with a frequency 8% [10]. However, there is still a significant proportion of NOA patients that have unknown etiology.

The establishment of in vitro fertilization using intracytoplasmic sperm injection (ICSI) as a standard treatment modality has resulted in a number of these men successfully fathering a child through surgically retrieved sperm from the testis. However, a genetic risk exists for these offspring, implying the necessity for future parents to be appropriately informed on potential consequences [11-13].

The aim of this study was to determine the prevalence of various chromosomal aberrations and the prevalence of Y-chromosome microdeletions among non-obstructive azoospermia Tunisian men attending the Pasteur Institute of Tunis.

Patients and Methods


The cytogenetic analysis has been focused on 401 Tunisian infertile patients with idiopathic non-obstructive azoospermia. These infertile men with sperm disorders were referred for karyotyping to the department of Histology and Cytogenetics at the Pasteur Institute of Tunis between January 2006 and May 2014. Among these patients, 90 non-obstructive azoospermia with normal karyotype were benefited from molecular analysis.

Patients were checked for the history of relevant medical disorders, e.g., diabetes, renal, liver disease, radiation, endocrine abnormalities (e.g., hypogonadotropic hypogonadism), exposure to toxins and/or medical affecting spermatogenesis, acquired and congenital structural defect of urogenital system, history of surgical intervention of genital tract.

Informed consent was obtained for karyotype and a molecular investigation from individual participants included in the study, and approval was given by the local ethics committee of Pasteur Institute of Tunis.


Cytogenetic analysis was performed from phytohemagglutininstimulated lymphocyte cultures by routine laboratory protocol. For microscopic analysis, R-banded metaphase spreads were analyzed and abnormalities recorded according to the current International System for Human Cytogenetic Nomenclature [14]. A resolution of 550 to 700 bands per haploid karyotype was used for the routine analysis. For each patient, at least 20 well-spread metaphases were analyzed and two to five metaphases were karyotyped. When at least one of the 20 showed a loss or gain of a chromosome, especially X or Y chromosome, the number of analyzed metaphases was increased to 30. If a second abnormal cell was observed, the analysis was considered complete; otherwise, the number of metaphases was increased to 50. Sex chromosome mosaics occurring at a level of less 5% were not considered as well as pericentric inversions of chromosome 9 or other structural chromosome variants and polymorphisms that were considered as normal cytogenetic events.

Detection of AZF microdeletions by multiplex PCR

Genomic DNA was extracted from blood lymphocytes using a commercially available kit (FlexiGene Kit; Qiagen), according to the manufacturer’s instructions. Each patient was examined for six AZF loci. The STS primers used were: for AZFa sY84 and sY86, for AZFb sY127 and sY134 and for AZFc sY254 and sY255. This primer set was suggested by Simoni et al. [9] and is prescribed by the European Academy of Andrology (EAA) and European Molecular Genetics Quality Network (EMQN) [15, 16]. In addition, sY14 (STS within the SRY gene located in Yp) was tested as an internal positive control. Two multiplex PCRs were carried out in 50 μl reaction volumes containing: 200 ng of each DNA sample, 1.5mM MgCl2, 0.4 mM of each dNTP, 1.6 μM of each oligonucleotide primer: sY86, sY127, sY254 (Mix A) for the first multiplex PCR, sY84, sY134, sY255 (Mix II) for the second one, 5% dimethyl sulfoxide (DMSO) were added to 1 μl of Taq DNA polymerase. A positive control (sample from a normal fertile male), and two negative controls [(i) normal female sample, (ii) every constituent except DNA], were included in every PCR assay. The reaction mixture included thermo cycling consisted of an initial denaturation of 5 minutes to 94°C followed by 35 cycles of 30 seconds at 94°C, 30 seconds annealing at 59°°C, 30 seconds extension at 720C, finally, 7 minutes extension step at 72°C. PCR products were analyzed by electrophoresis on 3% agarose gels containing ethidium bromide and visualized by exposure to ultraviolet light. In the event of detecting deletion with a primer, the PCR assay was repeated thrice for confirmation. A STS was considered absent only after 3 amplification failures in the presence of successful amplification of internal control (SRY).


Cytogenetic analysis

The average age was 38.07 ± 5.96 and the average duration of infertility was 5.26 ± 4.47 for the azoospermic patients.

Among these 401 patients, 49 cases showed abnormal karyotypes to a prevalence of 12.22%, in which 42 (10.47%) were sex chromosomal abnormalities and 7 (1.75%) were autosomal abnormalities.

The most common abnormality observed was the 47, XXY karyotype or their variant (mosaic 47, XXY/46, XY, 47, XXY/48, XXXY) consistent with KS (Figure 1), which were found in thirty seven cases (9.23%). The other chromosomal abnormalities was represented by: (6) balanced autosomal rearrangements, (2) 46, XX males (Figure 2), (2) unbalanced rearrangements, (1) 47, XYY (Figure 3), and (1) Yq deletion (Figure 4), (Table 1).